Most black flies of medical importance, including vectors of human onchocerciasis, are complexes of sibling species which can not be distinguished in all life stages. The long-term goal of this project is to develop rapid and accurate diagnostic techniques equally applicable to all life stages. Such an accomplishment would permit ecological studies leading to cost-effective and environmentally sound management practices targeted against only pestiferous siblings. It is thought that mitochondrial DNA (mtDNA) will provide such taxonomic characters but consideration is also to be given regions of the nuclear genome. Specific hypotheses to be tested are: (1) that species now recognized only by chromosomal differences in larvae, or by morphological characters applicable to a single life stage, can be identified in any life stage by mtDNA; and (2) that sequence divergence in mitochondrial genomes will be consistent with current classification at the species level. At least 3 presumed valid species in each of 3 species groups in Simulium (subgenera Psilozia and Psilopelmia, S. jenningsi group) will be examined. For at least one species in each group, field collections will be made over the range of the species to determine geographic variation. These populations will be examined morphologically, chromosomally, and mitochondrial genomes determined. Restriction maps of mitochondrial genomes will be constructed and compared to identify species-specific restriction sites. The identification of such sites will permit the design of identification protocols which utilize the amplification of diagnostic restriction fragments by Polymerase Chain Reaction (PCR), digestion of the amplified product with the appropriate restriction enzymes, and subsequent evaluation by gel electrophoresis. Consideration will also be given nuclear DNA by utilization of PCR random primers and hybridization with "simple sequence" probes which may provide more characters for closely related species.